Screening of significant biomarkers related with prognosis of liver cancer by lncRNA-associated ceRNAs analysis.

This study aimed to identify significant biomarkers related to the prognosis of liver cancer using long noncoding RNA (lncRNA)-associated competing endogenous RNAs (ceRNAs) analysis. Differentially expressed mRNA and lncRNAs between liver cancer and paracancerous tissues were screened, and the functions of these mRNAs were predicted by gene ontology and pathway enrichment analyses. A ceRNA network consisting of differentially expressed mRNAs and lncRNAs was constructed. LncRNA FENDRR and lncRNA HAND2-AS1 were hub nodes in the ceRNA network. A risk score assessment model consisting of eight genes (PDE2A, ESR1, FBLN5, ALDH8A1, AKR1D1, EHHADH, ADRA1A, and GNE) associated with prognosis were developed. Multivariate Cox regression suggested that both pathologic_T and risk group could be regarded as independent prognostic factors. Furthermore, a nomogram model consisting of pathologic_T and risk group showed a good prediction ability for predicting the survival rate of liver cancer patients. The nomogram model consisting of pathologic_T and a risk score assessment model could be regarded as an independent factor for predicting prognosis of liver cancer.

The expression and clinical significance of Omi/Htra2 in hepatocellular carcinoma.

BACKGROUND/AIMS: To investigate Omi/HtrA2 expression and its clinical significance in hepatocellular carcinoma. METHODOLOGY: We analyzed Omi/HtrA2 expression in hepatocellular carcinoma, paracancerous tissues and normal hepatic tissues by immunohistochemistry, RT-PCR and Western blot. Hypoxia-inducible factor-1α (HIF-1α) expression was also detected in hepatocellular carcinoma samples by immunohistochemistry. Meanwhile, we retrospectively analyzed the relationship between Omi/HtrA2 expression and the survival times of the patients. RESULTS: We found that Omi/HtrA2 overexpressed in hepatocellular carcinoma and was correlated with hepatocellular carcinoma differentiation, tumor size, portal vein invasion, clinical stage and lymph node metastasis. We also observed a significant inverse correlation between the expression of Omi,/HtrA2 and HIF-1α in hepatocellular carcinoma. The Kaplan-Meier estimates showed that patients who were Omi/HtrA2 positive had much longer survival times than those who were Omi/HtrA2 negative. Both univariate and multivariate analysis using the Cox regression model indicated that Omi/HtrA2 expression was a significant factor for prognosis. CONCLUSIONS: Hepatocellular carcinoma cells may need Omi/HtrA2 expression for apoptosis and Omi/HtrA2 might be an important prognostic marker for primary hepatocellular carcinoma.

Hypoxia-inducible factor-1alpha suppressed hepatocellular carcinoma cell apoptosis through influencing on Omi/HtrA2 expression and its releasing from the mitochondrion.

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays an important role in regulating hepatoma cell apoptosis. However, conclusions of different studies about the effects of HIF-1alpha expression on hepatoma cell apoptosis remain controversial. Omi/HtrA2 promotes cell apoptosis in some human cancer cells. Our previous experiments have demonstrated that primary hepatocellular carcinoma may need Omi/HtrA2 expression for cell apoptosis. Thus, we investigated the effect of HIF-1alpha on hepatocellular carcinoma cell apoptosis and Omi/ HtrA2 expression. In our study we found that HIF-1alpha gene could suppress hepatoma cell apoptosis, and Omi/HtrA2 mRNA and protein expression decreased with HIF-1alpha expression increase while Bcl-2 mRNA and protein expression increased with HIF-1alpha expression increase in HepG2 cells under normoxia condition. Meanwhile, Omi/HtrA2 protein expression increased with HIF-1alpha expression decrease in HepG2 cells under hypoxia culture. Taken together, these results demonstrated that HIF-1alpha suppressed hepatocellular carcinoma cell apoptosis through inhibiting Omi/HtrA2 expression and upregulating Bcl-2 expression to impede Omi/ HtrA2 releasing from the mitochondrion. The present finding further enriched and supported the role of HIF-1alpha expression on cell apoptosis of hepatoma cells.

[The prognostic significance of Omi/HtrA2 expression, and correlation between Omi/HtrA2 and hypoxia-inducible factor-1α in primary hepatocellular carcinoma cells].

OBJECTIVES: To investigate the prognostic significance of Omi/HtrA2 expression, and the correlation between Omi/HtrA2 and Hypoxia-inducible factor (HIF)-1α in primary hepatocellular carcinoma cells. METHODS: The expression of HIF-1α and Omi/HtrA2 in 43 cases of hepatic carcinoma tissues were detected immunohistochemically. Follow-up data were obtained to perform the Kaplan-Meier survival analysis. The change of Omi/HtrA2 expression in HepG2 cell was measured after HIF-1α expression of HepG2 in vitro was regulated by Tet-on expression system. RESULTS: Omi/HtrA2 expression was correlated with lymph node metastasis and recurring within liver during 2 years. Statistical analysis estimation showed the cumulative survival rate of post-hepatectomy for the patients with the positive expression of Omi/HtrA2 was higher than that for other patients with the negative expression of Omi/HtrA2 (χ(2) = 6.13, P = 0.013). In the common paraffin-embedded specimen of hepatocellular carcinoma, most of the samples showing negative or weak positive HIF-1α immunopositivity showed moderate positive or strong positive Omi/HtrA2 immunopositivity, while most of the samples showing moderate positive or strong positive HIF-1α immunopositivity showed negative or weak positive Omi/HtrA2 immunopositivity. The mRNA expression intensity of Omi/HtrA2 was decreasing with the HIF-1α expression increasing, and the difference was statistically significant(F = 106.766, P < 0.01). CONCLUSIONS: Omi/HtrA2 may be an important prognostic marker for primary hepatocellular carcinoma. Omi/HtrA2 expression is reversely correlated with HIF-1α expression in hepatocellular carcinoma. During the apoptotic process Omi/HtrA2 participating in hepatocellular carcinoma cells, HIF-1α is involved in the controlling and regulating of Omi/HtrA2 expression.

A SOX9-AS1/miR-5590-3p/SOX9 positive feedback loop drives tumor growth and metastasis in hepatocellular carcinoma through the Wnt/ß-catenin pathway.

Hepatocellular carcinoma (HCC) is a prevalent solid tumor with a high global death rate. SRY box 9 (SOX9) has been reported as an oncogene in HCC by several studies, but the underlying mechanism remains largely unexplored. Here, we confirmed upregulation of SOX9 in HCC tissues and cell lines and validated that SOX9 facilitates proliferation, migration and invasion in HCC. We subsequently identified that the long non-coding RNA (lncRNA) SOX9 antisense RNA 1 (SOX9-AS1) is a neighbor gene to SOX9; SOX9-AS1 is also upregulated in HCC, and its expression is positively correlated with that of SOX9. In addition, SOX9-AS1 appears to have prognostic significance in HCC patients. We showed that SOX9-AS1 aggravates HCC progression and metastasis in vitro and in vivo. We demonstrated that SOX9-AS1 sponges miR-5590-3p to elevate SOX9 expression, and that SOX9 in turn transcriptionally activates SOX9-AS1. Moreover, we verified that SOX9-AS1 regulates SOX9 and its known downstream Wnt/ß-catenin pathway so as to facilitate epithelial-to-mesenchymal transition. The results of our rescue assays suggest that SOX9-AS1 regulates HCC progression through SOX9 and the Wnt/ß-catenin pathway. In conclusion, our study demonstrates that a SOX9-AS1/miR-5590-3p/SOX9 positive feedback loop drives tumor growth and metastasis in HCC through the Wnt/ß-catenin pathway, suggesting SOX9-AS1 as a novel potential prognostic and treatment target for HCC.

Integrin αvß6 plays a bi-directional regulation role between colon cancer cells and cancer-associated fibroblasts.

Tumor microenvironment (TME) is the cellular environment in which tumor exists, and it contributes to tumor formation and progression. The TME is composed of tumor cells, stromal cells, cytokines, and chemotactic factors of which fibroblasts are the main cellular components. In our present study, we found that colorectal cancer (CRC) cells expressing integrin αvß6 clearly could induce morphological changes in inactive fibroblasts and increased the expression of activated fibroblast markers such as α-smooth muscle actin (α-SMA) and fibroblast-activating protein (FAP). Those activated fibroblasts in the TME are called cancer-associated fibroblasts (CAFs). In order to investigate the mechanism by which CRC cells expressing integrin αvß6 activated CAFs, a series of assays have been carried out in the follow-up. We found that CRC cells could secrete inactive transforming growth factor ß (TGF-ß); however, integrin αvß6 activated TGF-ß, which subsequently activated fibroblasts. This process was disrupted by knockdown of integrin αvß6. In contrast, activated fibroblasts could promote CRC cell invasion. In particular, the strengthening effect on expression of integrin αvß6 in colon cancer cells was obvious. Additionally, we found that CAFs could secrete stromal cell-derived factor-1 (SDF-1) and promote CRC cell metastasis in distant organs via the SDF-1/C-X-C chemokine receptor type 4 (CXCR4) axis. Taken together, we assumed that CRC cells and CAFs activated one another and worked together to promote cancer progression, with integrin αvß6 playing a role in the bi-directional regulation of these cells. Hence, integrin αvß6 may serve as a therapeutic target for the future CRC treatment.

The roles of four multi-drug resistance proteins in hepatocellular carcinoma multidrug resistance.

The roles of multi-drug resistance protein 1 (MDR1), multi-drug resistance related protein 1 (MRP1), lung resistance protein (LRP) and breast cancer resistance protein (BCRP) in the multi-drug resistance (MDR) of hepatocellular carcinoma (HCC) were studied. By exposing HepG2 cell line to progressively increased concentrations of adriamycin (ADM), HepG2 multi-drug resistant subline (HepG2/ADM) was induced. The MDR index of HepG2/ADM was detected by using MTT. The expressions of the four MDR proteins in the three cell lines (L02, HepG2, HepG2/ADM) were investigated at mRNA and protein levels by real-time RT-PCR and Western blot respectively. Our results showed that when the ADM concentration was under 100 microg/L, HepG2 could easily be induced to be drug-resistant. The IC(50) of the HepG2/ADM to ADM was 282 times that of HepG2. The expression of MDR1 and BCRP mRNA in HepG2/ADM cells were 400 and 9 times that of HepG2 cells respectively while there was no difference in the mRNA expressions of MRP1 and LRP. There was no difference between HepG2 and L02 cells in the mRNA expressions of the four genes. At the protein level, the expressions of MDR1, BCRP and LRP but MRP1 in HepG2/ADM were significantly higher than those of HepG2 and L02. Between HepG2 and L02, there was no difference in the expressions of four genes at the protein level. HepG2/ADM is a good model for the study of MDR. The four genes are probably the normally expressed gene in liver. The expressions of MDR1 and BCRP could be up-regulated by anti-cancer agents in vitro. The MDR of HCC was mainly due to the up-regulation of MDR1 and BCRP but MRP1 and LRP. These findings suggest they may serve as targets for the reversal of MDR of HCC.

[Inducible expression of hypoxia-inducible factor 1alpha on the proliferation and invasion property of HepG2 cells under normoxia in vitro].

OBJECTIVE: To study the inducible expression of hypoxia-inducible factor 1alpha (HIF-1alpha) on the proliferation and invasion property of HepG2 cells under normoxia in vitro. METHODS: Constructed the HepG2(Tet-on-HIF-1alpha) cell line which could induce the expression of HIF-1alpha by doxycycline; Under normoxia in vitro, MTT assay was used to observe the proliferative and adhesive activity of cells, and the invasive activity was determined by transwell cell culture chamber method. RESULTS: Under normoxia, the HIF-1alpha mRNA and protein of HepG2(Tet-on-HIF-1alpha) cells could be induced up to (5.899 +/- 2.176) and (2.179 +/- 0.742) folds by doxycycline (1 microg/ml); There were no difference of A(490 nm) between the Dox(+)and Dox(-) group in experiment detecting the proliferation activity (P > 0.05); But in adhesive experiment, the A(490 nm) of Dox (+) group was 0.662 +/- 0.058, higher than the Dox(-) group 0.526 +/- 0.808 (P = 0.008); The invasive cell number of Dox(+) group was 37.611 +/- 8.424, but in the Dox(-) group, the number was 25.333 +/- 8.117 (P < 0.01). CONCLUSIONS: Under normoxia in vitro, the Tet-on gene regulate system could increase the HIF-1alpha protein by inducing the HIF-1alpha mRNA; HIF-1alpha has no influence with the proliferation activity, but it could enhance the adhesive and invasive properties of HepG2 cells.

[Expression of Twist gene in human hepatocellular carcinoma and its clinicopathological significance].

OBJECTIVE: To investigate the expression and clinicopathological significance of transcription factor Twist in hepatocellular carcinoma (HCC), paraneoplastic and cirrhotic tissues. METHOD: Immunohistochemistry was used to detect the expression of the Twist protein in 26 cases of HCC and paraneoplastic tissue and 10 cases of cirrhotic tissue. Meanwhile, the Twist mRNA and its protein were detected in 10 HCC tissues and 10 paraneoplastic tissues by RT-PCR and Western blot. And the expression differences and clinicopathological significances of the expression of Twist gene and its protein were analyzed. RESULTS: The positive rates of the Twist protein in HCC, paraneoplastic and cirrhotic tissues were 84.6%, 19.2 % and 20.0 %, respectively. The positive rate of Twist in HCC was higher than that in paraneoplastic or cirrhosis tissues (P < 0.05). Compared with in paraneoplastic tissues, the mRNA and protein expression of Twist were up-regulated in HCC by 2.52 and 2.13 times, respectively. CONCLUSION: Twist has an inappropriate expression in hepatocellular carcinoma and it may play an important role in the tumorigenesis and progression of HCC.

Norcantharidin Suppresses Colon Cancer Cell Epithelial-Mesenchymal Transition by Inhibiting the αvß6-ERK-Ets1 Signaling Pathway.

Norcantharidin (NCTD) is an efficacious anti-cancer drug that has been used in China for many years, but its underlying mechanism of action is still not fully understood. In the present study, we found that NCTD could induce morphological changes in colon cancer cells, causing a transition from a spindle-shaped morphology to a typical round or oval shape, which was indicative of a mesenchymal-epithelial transition (MET) process. Next, we investigated the mechanism by which NCTD induced the MET process. Using a transwell assay, we found that NCTD could suppress the migratory and invasive ability of colon cancer cells in a dose-dependent manner. Moreover, NCTD suppressed the expression of integrin αvß6, MMP-3, and MMP-9 as well as the polymerization of F-actin, further supporting its suppressive effect on migratory and invasive ability. Furthermore, the expression of αvß6, N-cadherin, vimentin and phosphorylated ERK was decreased, while the expression of E-cadherin was up-regulated. We verified that phosphorylated Ets1 was down-regulated substantially after treatment with NCTD. Taken together, our data demonstrated that NCTD could inhibit the EMT process of colon cancer cells by inhibiting the αvß6-ERK-Ets1 signaling pathway. This study revealed part of the mechanism through which NCTD could reverse the EMT process in colon cancer.

Transcatheter arterial chemoembolization as an examination method for hepatocellular carcinoma undetected by B-mode ultrasound, computed tomography and digital subtratcion angiography: A case report.

Transcatheter arterial chemoembolization (TACE) is the conventional treatment for patients with unresectable hepatocellular carcinoma (HCC), but few studies to date have demonstrated the use of TACE as an examination method for uneasily detected HCC. The present study describes an unusual case of HCC with TACE as an examination method. A 41-year-old male presented with an elevated α-fetoprotein level (AFP) of 3,635 ng/ml, however, no tumor lesions were detected by B-mode ultrasound, computed tomography (CT) or digital subtraction angiography. During TACE treatment, two tumor lesions of ~0.5 and 0.8 cm were revealed in the right liver lobe, with no tumors in the left liver lobe. A month after TACE, a liver CT scan found 11 lesions (8 in the right liver lobe and 3 in the left liver lobe). The HCC patient's AFP levels decreased to an almost normal level following the TACE treatment. This study provokes consideration of the application of TACE in the diagnosis and treatment of HCC patients with liver lesions that are hard to detect by conventional means.

Natural Orifice Total Transtracheal Endoscopic Thyroidectomy Surgery: First Reported Experiment.

BACKGROUND: Natural orifice translumenal endoscopic surgery (NOTES(®); American Society for Gastrointestinal Endoscopy [Oak Brook, IL] and Society of American Gastrointestinal and Endoscopic Surgeons [Los Angeles, CA]) is an improvement in surgical interventions. In this study we developed an innovative transtracheal endoscopic thyroidectomy technique and explored its feasibility in animal models. MATERIALS AND METHODS: Transtracheal endoscopic thyroidectomy was performed in anesthetized dogs and pigs. The endoscope was advanced into the pretracheal space via a longitudinal incision on the anterior tracheal wall. Hemithyroidectomies and partial lobectomy were performed using special double-lumen endotracheal tubes and conventional endoscopic instruments. The tracheal wall incision was closed using absorbable sutures, and the animals were sacrificed at Day 5 postsurgery. RESULTS: Hemithyroidectomy and partial thyroidectomy were successfully performed on pigs and dogs. The average operative time for each model was 69.4 minutes. No significant complications were encountered during surgery. CONCLUSIONS: The transtracheal endoscopic thyroidectomy technique is feasible and has the potential to be an alternative method for other types of thyroid surgeries.

Special AT-rich sequence-binding protein-1 participates in the maintenance of breast cancer stem cells through regulation of the Notch signaling pathway and expression of Snail1 and Twist1.

The stem cell populations in cancerous tissues and cell lines vary widely and are often associated with aggressive cases of breast cancer. Despite research on the topic, the mechanism underlying the regulation of the breast cancer stem cell (BCSC) population within tumors remains to be fully elucidated. To investigate the function of special AT­rich sequence­binding protein­1 (SATB1) in the maintenance of the BCSC population, SATB1 was overexpressed with lentivirus in MCF­7 cells or knocked down with shRNA­lentivirus in BT­549 cells. The effects of SATB1 overexpression or knockdown on mammosphere formation, the size of the of BCSC population, cell invasion and tumorigenesis were investigated. Activation of the Notch signaling pathway and expression of Snail1 and Twist1 were also examined in the cells. Overexpression of SATB1 in MCF­7 cells was observed to increase mammosphere formation, the size of the BCSC population, cell invasion and tumorigenesis, accompanied by an increase in the activation of Notch signaling and expression levels of Snail1 and Twist1. Conversely, knockdown of SATB1 in BT­549 cells produced the opposite effects. The results indicated that expression of SATB1 may increase the size of the BCSC population via the activation of the Notch signaling pathway and by increasing expression levels of Snail1 and Twist1.

Omi/HtrA2 pro-apoptotic marker differs in various hepatocellular carcinoma cell lines owing to ped/pea-15 expression level.

Omi/HtrA2 promotes cell apoptosis in human cancer cells. Early studies showed that primary hepatocellular carcinoma requires Omi/HtrA2 expression for cell apoptosis. Additionally, the Omi/HtrA2 pro-apoptotic marker demonstrated a difference in some cell types. However, how the Omi/HtrA2 pro-apoptotic marker reacts during the process of hepatocellular carcinoma cell apoptosis remains to be determined. Thus, we investigated the role and possible mechanism of Omi/HtrA2 on hepatocellular carcinoma cell apoptosis using various hepatocellular carcinoma cell lines. The results were analyzed using RT­qPCR and western blot analysis. In the present study, we found that Omi/HtrA2 was overexpressed in hepatocellular carcinoma cell lines and induced hepatocellular carcinoma cell apoptosis. Additiionally, the only manner in which Omi/HtrA2 participated in cell death in PLC cells may be dependent on IAP-binding. Omi/HtrA2­inducing HepG2 cell apoptosis may mainly depend on its serine protease activity while both IAP-binding and its serine protease activity participated in Hep3B cell apoptosis. This result suggested that Omi/HtrA2 pro-apoptotic marker differs in various hepatocellular carcinoma cell lines. PLC cells were also devoid of the expression of ped/pea-15 as the substrate of Omi/HtrA2 serine protease while ped/pea-15 was overexpressed in HepG2 and Hep3B cells and ped/pea-15 expression was higher in HepG2 cells than that in Hep3B cells. These results showed that Omi/HtrA2 overexpression promotes hepatocellular carcinoma cell apoptosis and the ped/pea-15 expression level causes this difference of the Omi/HtrA2 pro-apoptotic marker in the various hepatocellular carcinoma cell lines.

Clinical and pathological features and surgical treatment of Budd-Chiari syndrome-associated hepatocellular carcinoma.

BACKGROUND: Budd-Chiari syndrome (BCS) is characterized by liver sinusoidal congestion, ischemic liver cell damage, and liver portal hypertension caused by hepatic venous outflow constriction. The aim of this research was to investigate the clinicopathological features of BCS-associated hepatocellular carcinoma (HCC) and explore its surgical treatment and prognosis. METHODS: Clinical data from 38 patients with BCS-associated HCC who were surgically treated in our hospital from July 1998 to August 2010 were retrospectively analyzed. The clinicopathological features and prognosis of patients with BCSassociated HCC and surgical treatment for BCS-associated HCC were investigated. RESULTS: Compared to the patients with hepatitis B virus (HBV)-associated HCC, the patients with BCS-associated HCC showed a female predominance, and had significantly higher cirrhosis rate, higher incidence of solitary tumors, lower incidence of infiltrative growth, higher proportion of marginal or exogenous growth, lower rate of portal vein invasion, and higher degree of differentiation. Median survival was longer in patients with BCS-associated HCC (76 months) than in those with HBV associated HCC (38 months). Of 38 patients with BCS-associated HCC, 22 patients who received combined surgery mainly by liver resection plus cavoatrial shunts exhibited hepatic venous outflow constriction relief, while the other 16 patients only underwent liver resection. The combined surgery group had significantly longer survival and lower incidences of post-operative lethal complications (P < 0.05). Multivariate analysis showed that relief of hepatic venous outflow obstruction was a protective factor for survival of patients with BCS-associated HCC, whereas portal vein invasion was a risk factor. CONCLUSIONS: BCS-associated HCC has a more favorable biological behavior and prognosis than HBV-associated HCC. For patients with BCS-associated HCC, tumor resection accompanied with relief of hepatic venous outflow obstruction can reduce the incidence of complications and extend survival.

Role of hypoxia-inducible-1α in hepatocellular carcinoma cells using a Tet-on inducible system to regulate its expression in vitro.

Hypoxia-inducible-1α (HIF-1α) expression was intimately correlated with apoptosis and proliferation of cancer cells. However, conclusions of different studies on the effects of HIF-1α expression on cell apoptosis and cell proliferation of hepatoma cells remain controversial. In view of the current status, we reassess its roles and possible mechanism in hepatoma cells. In order to acquire more convincing and reliable results, we used a Tet-on system to stably and effectively regulate HIF-1α expression in the HepG2 cells in vitro. In our study we not only confirmed some common conclusions of previous studies, but also acquired some different and significant results that HIF-1α facilitates cell proliferation and cell cycle through influencing the expression of cyclin A and cyclin D, and suppresses cell apoptosis through inducing the expression of survivin and Bcl-2. These results further enrich our knowledge on the role of HIF-1α expression on cell apoptosis and cell proliferation of hepatoma cells.